Pyruvate ester composition and method of use for resuscitation after events of ischemia and reperfusion

ABSTRACT

A therapeutic composition comprising an alkyl, aralkyl, alkoxyalkyl or carboxyalkyl ester of 2ketoalkanoic acid and a component for inducing and stabilizing the enol resonance form of the ester at physiological pH values is disclosed. The composition of the invention further comprises a pharmaceutically acceptable carier vehicle in which the enol resonance form of the ester is stabilized at physiological pH values. Formulations containing the compositions of the invention permit the successful use of 2-ketoalkanoic acid esters, e.g., pyruvic acid esters, to treat, e.g., is chemic events, shock, organ reanimation, resuscitation and other recognized pyruvate-effective treatments. The compositions of the inventions are also useful in a process for preserving organ parts, organs or limbs removed from a living mammal and in need of preservation, e.g., for later transplantation to an organ recipient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/116,707, filed Apr. 4, 2002, which is a continuation of InternationalApplication No. PCT/US00/27758, which designated the United States andwas filed on Oct. 6, 2000, published in English, which claims thebenefit of U.S. Provisional Application No. 60/158,091, filed on Oct. 7,1999. The entire teachings of the above applications are incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Part of the work leading to this invention was carried out with UnitedStates Government support provided under a grant from the HationalInstitutes of Health, Grant No. GM3763 1. Therefore, the U.S. Governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

This invention relates to several new pyruvate compounds and methods forresuscitation and reanimation of mammals, especially humans, before,during and after, e.g., (1) mesenteric ischemia, mesenteric thrombus ormesenteric venous occlusion; (2) aortic aneurism repair, coronary arterybypass, surgical treatment of arterial occlusion of limbs; (3)hemorrhagic shock, resulting from either penetrating and blunt trauma;and (4) preservation and transplantation of organs. Ischemia is definedherein as the interruption of oxygen supply, via the blood, to an organor to part of an organ. Examples of ischemic events include (i)myocardial, cerebral, or intestinal infarction following obstruction ofa branch of a coronary, cerebral, or mesenteric artery, and (ii) removaland storage of an organ prior to transplantation. In the case ofmyocardial infarction, prompt restoration of blood flow to the ischemicmyocardium, i.e. coronary reperfusion, is a key component of thetreatment. This is because mortality is directly related to infarct size(tissue necrosed) which is related to the severity and duration of theischemic event. The consequences of hemorrhagic shock are similar tothose of ischemia, although the causative event is not an interruptionof blood flow but rather the event of massive blood loss itself whichcauses deprivation of the oxygen supply.

Notwithstanding the need to supply an organ cut-off from a normal bloodsupply with oxygen, it has been found that reperfusion injury may occurupon restoration of blood flow. This results from the production ofreactive oxygen species (ROS), namely, hydrogen peroxide, hydroxylradicals and superoxide radicals, among others, which are formed fromboth extracellular and intracellular sources. ROS are highly reactivespecies that, under normal conditions, are scavenged by endogenousdefense mechanisms. However, under conditions of post-ischemic oxidativestress, ROS interact with a variety of cellular components, causingperoxidation of lipids, denaturation of proteins, and interstitialmatrix damage and resulting in increase of membrane permeability andrelease of tissue enzymes.

In an attempt to minimize these undesirable side effects of perfusion inthe treatment of ischemia and also of shock, researchers havedemonstrated the utility of various antioxidants in the reperfusionprocess.

Banda et al. (1996), together with Kurose et al. (1997), suggested theuse of an inhibitor of ROS production to protect the reperfusedmyocardium and the use of agents and inhibitors that reduce ROS levels.In a similar context, desiring to provide more efficient resuscitation,researchers have demonstrated the additive utility of incorporating anantioxidant and a beneficial metabolic fuel into the reperfusionregimen. Salahudeen et al. (1991) used solutions of pyruvate, an ROSscavenger and a metabolically important precursor fuel forgluconeogenesis, to protect against hydrogen peroxide induced acuterenal failure. Cicalese et al. (1996) found that pretreatment withintraluminal pyruvate ameliorates post ischemic small bowel injury whileCrestanello et al. (1998), DeBoer et al. (1993), and O'Donnell-Tormey etal. (1987) have substantiated this finding by examining the ameliorativeeffects of both endogenously secreted pyruvate and exogenously addedmaterial in the reperfusion and subsequent function of organ and tissuepreparations subjected to ischemia and simulated shock. Varma et al.(1998), similarly, have shown that in a cultured lens system, afterexposure of the cultured lens to free radical oxidant stress, pyruvateand its esters have certain cytoprotecting and restorative effects.

In a further effort directed to protecting reperfused heart tissue, U.S.Pat. No. 5,075,210, herein incorporated by reference, discloses aprocess for reperfusing a heart for transplantation. The patentdiscloses a cardioplegic solution containing sodium chloride, potassiumchloride, calcium chloride, sodium bicarbonate, sodium EDTA, magnesiumchloride, sodium pyruvate and a protein.

U.S. Pat. No. 5,294,641, herein incorporated by reference, is directedto the use of pyruvate to prevent the adverse effects of ischemia. Thepyruvate is administered prior to a surgical procedure to increase apatient's cardiac output and heart stroke volume. The pyruvate isadministered as a calcium or sodium salt. The pyruvate can alternativelybe an amide of pyruvic acid such as ethylamino pyruvate. Similarly, U.S.Pat. No. 5,508,308, herein incorporated by reference, discloses the useof pyruvyl glycine to treat reperfusion injury following myocardialinfarction.

U.S. Pat. Nos. 4,988,515 and 5,075,210, herein incorporated byreference, use pyruvate salts in cardioplegic solutions and inpreservation solutions for the heart before transplantation. U.S. Pat.No. 4,970,143, herein incorporated by reference, discloses the use ofacetoacetate for preserving living tissue, including addition of thepyruvate to the preservation solution.

U.S. Pat. No. 5,100,677 herein incorporated by reference, discloses thecomposition of various parenteral solutions. Of interest is arecommendation to include pyruvate anions (apparently from metal salts)in intravenous solutions.

U.S. Pat. No. 5,798,388, herein incorporated by reference, furtherdescribes the utility of pyruvate salts and of various complexderivatives, such as amides, for the treatment of ROS in the context ofairway inflammation. The patent discloses a pyruvate compound in theform of a covalently linked pyruvoyl-amino acid. By utilizing this typeof a pyruvate delivery system, the negative effect of pyruvate salt isavoided. However, administration of large amounts of pyruvate-amino acidmay result in nitrogen overload which could harm patients with liverand/or kidney pathology.

In a similar context and based on a similar rationale for pyruvatedelivery, U.S. Pat. No. 5,876,916 pertains to the utility of pyruvatethiolesters and polyol esters for the treatment or prevention ofreperfusion injury following ischemia, diabetic effects, cholesterollevels, injured organs, ethanol intoxication or as a foodstuff; and U.S.Pat. Nos. 5,633,285; 5,648,380; 5,652,274; and 5,658,957, each hereinincorporated by reference, disclose various compositions, salts,prodrugs and derivatives of pyruvate in mixtures with otherantioxidants, fatty acids as anti-inflammatory and immunostimulatingwound healing compositions. However, administration of large amounts ofcomplex pyruvate-amino acid and other pro-drug derivatives requiringenzymatic hydrolysis prior to liberation of their antioxidant effectsmay result in nitrogen and/or other xenobiotic overload, which couldharm patients directly, interfere with normal detoxifying processes, orcause toxic effects through by-products of limited shelf life.

Notwithstanding the acceptance of pyruvate as an effective component ofa reperfusion solution or other varied applications, pyruvic acid is astrong and unstable acid which cannot be infused as such. On standing insolution, pyruvic acid and its salts at various pH values, including inthe physiological range, are known to form both a stable hydrate and adimer (para-pyruvate), neither of which reach with ROS as antioxidantsand both of which are known inhibitors of pyruvate utilization as ametabolic fuel, thereby abrogating any of the beneficial effects whichmight have accured from pyruvate administration in accordance with theprior art just described.

Furthermore, it has been recognized that traditional pharmacologicalpyruvate compounds, such as salts of pyruvic acid, are not particularlyphysiologically suitable. For example, these compounds lead to theaccumulation of large concentrations of ions (e.g., calcium or sodium)in the patient's body fluids. Similarly, amino acid compounds containingpyruvate can lead to excessive nitrogen loads. It has also been proposedto infuse pyruvylglycine, the amide function of which is presumablyhydrolyzed in plasma and/or tissues, thus liberating pyruvate.

However, at the high rates of pyruvoylglycine infusion required toachieve 1 mM pyruvate in plasma, the glycine load may be harmful topatients suffering from hepatic or renal pathologies. Also, floodingplasma with glycine may interfere with the transport of some amino acidsacross the blood-brain barrier. Accordingly, while potentially suitableto organ preservation, these pyruvate compounds are less suited totreating an organ in vivo, and it is recognized that a need exists toprovide a pyruvate delivery compound that is more physiologicallyacceptable.

There is also a recognized need to provide a pyruvate delivery systemthat is cost effective, simple, and devoid of opportunities forcontamination because of 1) limited shelf-life, 2) complexity offormulation, 3) reactivity and co-reactivity with excipients and otherformulation materials, 4) adverse biochemical reactivity duringtransport, translocation, and uptake into tissues, and 5) therequirement for metabolic activation via enzymatic hydrolysis byamidases or peptidases. Therefore, it would be desirable to haveavailable an alternate physiologically compatible therapeutic pyruvatecompound.

SUMMARY OF THE INVENTION

The invention described herein provides a new and improved, accessiblecomposition for the above-indicated uses.

In one aspect, the invention is directed to a composition comprising analkyl, aralkyl, alkoxyalkyl or carboxyalkyl ester of 2-ketoalkanoic acidand a component for inducing and stabilizing the enol resonance form ofthe ester at physiological pH values. The composition of the inventionfurther comprises a pharmceutically acceptable carier vehicle in whichthe enol resonance form of the ester is stabilized at physiological pHvalues.

Preferably, the ester in the composition of the invention is an alkylester of 2-ketopropionic acid (pyruvic acid), most preferably the ethylester, and the stabilizing component is a cationic material, preferablya divalent cation, and most preferably calcium or magnesium. Thepharmaceutically acceptable carrier in the composition of the inventioncan be any carrier vehicle generally recognized as safe foradministering a therapeutic agent to a mammal, e.g., a buffer solutionfor infusion, a tablet for oral administration or in gel, micelle orliposome form for on-site delivery. A preferred buffer solution isisotonic or hypertonic saline; or a bicarbonate, phosphate, plasmaextender, microcolloid or microcrystalline solution. Particularlypreferred is Ringer's solution of isotonic saline supplemented withpotassium ion. In a particularly preferred aspect, the composition ofthe invention comprises ethyl pyruvate admixed with calcium ion in aRinger's solution at a pH in the range of 7-8.

In other aspects, the ester portion of the 2-ketoalkanoic acid estercompound in the composition of the invention is selected preferably fromthe group consisting of ethyl, propyl, butyl, carboxymethyl,acetoxymethyl, carbethoxymethyl and ethoxymethyl esters. The2-ketoalkanoic acid portion is selected preferably from the groupconsisting of 2-keto-butyrate, 2-ketopentanoate,2-keto-3-methyl-butyrate, 2-keto-4-methyl-pentanoate and2-keto-hexanoate.

In another aspect, the invention is directed to methods for treatinginjuries, conditions or disorders associated with events such asischemic events or reperfusion. Formulations containing the novelcompositions of the invention permit the successful use of2-ketoalkanoic acid esters, e.g., pyruvic acid esters, to treat, e.g.,ischemic events, shock, organ reanimation, resuscitation and otherrecognized pyruvate-effective treatments as sufficiently high loads ofpyruvate can be administered without a toxic constituent. Moreover, useof the compositions of the invention provides a direct replacement fortraditional lactated Ringer's solutions uncomplicated by the addition ofco-active ingredients or complex excipients, such as those comprised ofmultiple compounds or molecular derivatives of pyruvate itself. Thecompositions of the inventions are also useful in a process forpreserving organ parts, organs or limbs removed from a living mammal andin need of preservation, e.g., for later transplantation to an organrecipient. Such processes are well known to those of skill in the art,e.g., as described in U.S. Pat. No. 5,066,578, hereby incorporated byreference herein.

A further practical advantage of the methods of the invention is theformulation of the active 2-ketoalkanoic acid ingredient as abiologically safe, readily hydrolyzable ester which can be taken up intotissues and cells by diffussive processes through membranes, owing tosaid ester's greater lipophilicity over the corresponding salt, whileretaining the ability to be hydrolyzed intracellularly by means ofnon-specific esterases and/or non-specific, marginally alkalinesolvolysis catalyzed by organic acids or bases such as amino acidresidues at physiological pH values.

More importantly, the method of this invention provides 2-ketoalkanoicacids, e.g., pyruvic acid, in a stabilized ester form that inactivatesreactive oxygen species by more than one mechanism of reaction and whosereaction products with reactive, hypervalent oxygen, such as hydrogenperoxide, affords degradation products that themselves are metabolicfuels instead of potentially harmful excretory products or metabolites.

BRIEF DESCRIPTION OF THE DRAWINGS

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims, taken in conjunction with the accompanying drawings, in which:

FIG. 1 shows the structures of the preferred 2-ketoalkanoic acid estersin the composition of the invention;

FIG. 2 shows the structures of certain preferred esters in thecomposition of the invention, their enol resonance structures and thestructures of certain prior art compounds;

FIG. 3 shows the system and computational parameters used for themeasurement of mucosal-to-serosal intestinal permeability followingpractice of the method of the invention;

FIG. 4 shows the intestinal permeability results achieved for a controlcomposition relative to compositions of the invention; and

FIG. 5 shows the results obtained for mucosal injury scores forcompositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, it is a primary object of this invention to provide new andimproved compositions containing 2-ketoalkanoic acid esters and methodsof using them to treat certain conditions as described above.

To achieve the foregoing objects and in accordance with the purpose ofthe invention, as embodied and broadly described herein, one novelcomposition of this invention comprises a 2-ketoalkanoic acid ester, inaccordance with the molecular structures shown in FIG. 1, admixed with asufficient concentration of biologically safe organic or inorganiccations to induce enolization of the 2-keto functionality of the esterat physiological pH values. In a preferred embodiment, the compositioncomprises an alkyl ester of 2-ketopropionic acid (pyruvic acid), theester is the ethyl analog and the cation is a divalent cation,particularly either calcium or magnesium. In a particularly preferredformulation of the composition of the invention, the ester compound isethyl pyruvate admixed with calcium ion in a Ringer's solution at a pHof about 7-8.

The therapeutic compositions of the invention may be administeredorally, topically, or parenterally, (e.g., intranasally, subcutaneously,intramuscularly, intravenously, intraluminally, intra-arterially,intravaginally, transurethrally or rectally) by routine methods inpharmaceutically acceptable inert carrier substances. For example, thetherapeutic compositions of the invention may be administered in asustained release formulation using a biodegradable biocompatiblepolymer, or by on-site delivery using micelles, gels, liposomes, or abuffer solution. The active ester agent in the composition of theinvention can be administered, as an infusate, at a concentration of,e.g., 20-200 mM, at a rate of, preferably, 10-100 mg/kg/hr, in a buffersolution as described herein. In bolus form, the active ester agent canbe administered at a dosage of, e.g., 10-200 mg/kg from 1-4 times daily.The cation in the composition of the invention is at an appropriateconcentration to induce enolization of the 2-keto functionality of theamount of active ester agent in the administered composition. Optimaldosage and modes of administration can readily be determined byconventional protocols.

It is believed that pyruvate, and other 2-ketoalkanoic acids, whenliberated intracellularly from the esters delivered, e.g., by thereanimation perfusate, acts as a NADH trap and a trap for ROS generatedupon reperfusion. In the first instance, a 2-ketoalkanoic acid reacts toafford lactate, oxidizing excess NADH and thereby protecting against the“reductant stress” generated during the physiological insult caused byhypoxia. In the latter instance, a 2-ketoalkanoic acid reacts withhypervalent oxygen, as demostrated in the prior art, to form a transientperacid which decomposes spontaneously, and eventually, to acetate andcarbon dioxide. The resulting acetate is a waste product, which may besalvaged by re-entry into the acetylCoA pool and harvested biochemicallyvia intermediary metabolism in the Krebs cycle or via gluconeogenesis.

However, and more significantly for the purposes of this invention, the2-ketoalkanoic acid ester itself serves as an antioxidant by a differentmechanism, namely, via reaction with hypervalent oxygen at the enolmethylene group. ROS is a membrane associated process, since hypervalentoxygen is generated by a redox cascade mediated by cytochromes in themicrosomes or the mitochodria. It is also an intracellular process thattakes place in lipophilic environment rather than in cytosol, and thethermodynamic properties of a 2-ketoalkanoic acid ester are such thatits reactivity towards redox reaction in a lipophilic phase isputatively favored by the cation mediated keto-enol equilibrium. Abinitio and semi-empirical thermodynamic analyses on ethyl pyruvate as arepresentative enolizable molecule in the presence of calcium arediscussed in greater detail as part of Example I below.

For example, using pyruvate as the exemplary 2-ketoalkanoic acid,formation of transient epoxides and subsequent rearrangement affords thecorresponding hydroxylated pyruvate esters at the 3-carbon, by amechanism similar to that of 3-hydroxy-pyruvate formation inintermediary metabolism as well as that of carbon additions to thephosphoenolpyruvate congener. Hydroxylation alpha to keto groups is alsoa recognized cytochrome mediated process in steroid metabolism and inmicrosomal hydroxylation of drugs. The resulting hydroxypyruvates, inturn, when solvolyzed into the carboxylic anions, can then react onceagain with hypervalent oxygen to afford hydroxyacetic acid (glycolicacid), the net result being that pyruvate esters can ultimately quenchtwo equivalents of ROS while pyruvates are limited thermodynamically toquenching only one. As mentioned above, 2-ketoalkanoic acid esters otherthan pyruvate esters are also appropriate for use in compositions of theinvention as long as the active compound is metabolizable as describedabove for the pyruvate ester.

The following examples are presented to illustrate the advantages of thepresent invention and to assist one of ordinary skill in making andusing the same. These examples are not intended in any way otherwise tolimit the scope of the disclosure.

EXAMPLE 1 Thermodynamic Modeling of Pyruvate Esters

Semiempirical quantum chemistry permits the comparative evaluation ofvarious pyruvate analogs with regard to the properties that determineeach molecule's reactivity. As one can note a marked difference in thebiological effect of ethyl pyruvate versus sodium pyruvate asantioxidants, the hypothesis that these two molecules arethermodynamically different can be tested by Huckel Molecular Orbital(HMO) analysis followed by Complete Neglect of Differential OverlapAnalysis (CNDO), using Molecular Modeling Pro/MOPAC software (ChemSW,Inc. Fairfield, Calif.). The following results were obtained for thestructures shown in FIG. 2, after their conformations were set by energyminimization to the optimal conformation: TABLE 1 Comparison ofThermodynamic Properties Compound Energy Dipole LogP H-Acceptor H-DonorNa Pyruvate (1) −31.7 355.6 −85.9 17.8 2.9 Na Pyruvate −16.5 462.8 −71.223.9 4.8 Hydrate (2) Na Enol-pyruvate −30.7 358.0 −72.1 17.7 2.8 (3)Ethyl pyruvate −86.5 2.8 −.21 .73 8.5 (4) Ethyl −84.1 2.5 −.37 .71 7.3enol-pyruvate (5) Ca enol ethyl −82.3 2.7 −.41 .85 7.2 pyruvate (6)

From the trend in minimization energies, the lower and, therefore, themore stable configurations are those associated with the pyruvateesters, although the differences all fall within an order magnitude. Onthe other hand, the esters show markedly lower dipole moments,reflecting their relatively weak ionization and dissociation potentials,a fact that is further supported by the higher LogP values, which are ameasure of relative lipophilicity. Also, the esters are poorer hydrogenbonding acceptors and better hydrogen bonding donors, consistent withtheir dipolar and lipophilic properties.

Thus, on an ab initio thermodynamic basis, one would predict that ethylpyruvate, and its putative partition enol tautomers, are more likely topartition between a polar aqueous phase and a lipid phase, whileretaining conformational stability of the same order as the pyruvatesodium salts. Further, it should be noted that the coordination complexof the pyruvate enolate ester with a divalent cation, such as calcium,shown in FIG. 2 as structure 6, affords the most pronounced change inproperties over pyruvate itself, substantiating the utility of thesecation-enolate-ester complexes as promoters of heretofore unexploitedreactivities of the pyruvate carbon skeleton conformation.

EXAMPLE 2 Reactivity Modeling of Pyruvate Esters

Searches of the Chemical Abstracts and the ISIS databases (MDLInformation Systems, Inc.) were conducted to uncover actual examples ofthe reactivity of pyruvates and their enolates. While numerousprecedents for the reactions of pyruvate salts have been recorded, farfewer examples of the molecular interactions between pyruvate esters andhypervalent oxygen are reported in the organic and biochemicalliterature. The principal reactions of pyruvates at physiological pHvalues are hydrate formation (FIG. 2, structure 2) and dimerization topara-pyruvate (FIG. 2, structure 7).

As reported by Margolis et al. (1986), sodium pyruvate at concentrationsof 1 Mol/liter or less forms varying amounts of the hydrate and thelinear dimer, 4-hydroxy-4-methyl-2-ketoglutaric acid. The hydrate canreach 6-10% and the dimer 20-25% on standing for 48 hrs. This reactivitypattern is an important consideration in the evaluation of sodiumpyruvate-containing infusates and perfusates, since the hydrate isunreactive towards hypervalent oxygen and the dimer is an inhibitor of2-ketoglutarate dehydrogenase, a mitochondrial respiratory enzyme, aswell as an inhibitor of glutamate transaminases and lactic aciddehydrogenase. By contrast, neither hydrate formation nor dimerizationof pyruvate esters have been reported in the chemical literature.

While the enol forms of pyruvate are thermodynamically stable inprinciple, their occurrence in aqueous media is unfavored and half-livesof enolates are measurable only in the 3-5 sec range (Kuo et al.(1979)). As the polarity of the solvent decreases, exemplified by thesolvation environment provided by dimethylsulfoxide ordimethylformamide, the half life of the enol increases by at least twoorders of magnitude (Chiang et al. (1993); Peliska et al. (1991); Sawyeret al. (1983).

As to reactivity toward hypervalent oxygen, both pyruvate salts andpyruvate esters react to form an initial hydroperoxide intermediate atthe carbonyl site, which rearranges by disproportionation to affordacetic acid and carbon dioxide or ethoxycarbonic acid, which undergoessubsequent aqueous solvolysis into carbon dioxide and ethanol(Constantopoulos et al (1984); Sawyer et al. (1983); Starostin et al.(1980)).

However, enolpyruvates can also react by an alternate mechanism thatinvolves addition to the exo-methylene group, as in the case ofenolpyruvate C-bromination at the 3-carbon (Sekine et al. (1980)), thechelation controlled addition to allylic compounds (Muderawan et al.(1998)), and the biological addition of carbon dioxide to formoxaloacetate via phosphoenolpyruvate carboxylase (Ausenhus et al.(1992)). Enols of biological ketones in general, as exemplified byD-ring acetyl steroids, react with activated oxygen via the cytochromeP-450 oxidase system to afford hydroxyketones via a transientexomethylene epoxide intermediate (Yamazaki et al. (1997)).

When evaluated on the grounds of thermodynamic likelihood and chemicalprecedent, pyruvate salts can be predicted, via the REACCS softwaredatabase correlation system, to react with hypervalent oxygen to affordonly decarboxylation to acetate and carbon dioxide. Pyruvate esters, onthe other hand, can be expected to afford not only the paireddecarboxylation products, acetate and alcohol, but also hydroxylatedadducts at the 3-carbon, most probably a 3-hydroxypyruvate. These latterspecies can again react with hypervalent oxygen to yield glycolic acidand carbon dioxide (Perera et al. (1997)), thereby consuming twoequivalents of oxidant.

EXAMPLE 3 Stability and Reactivity of Pyruvate Esters in Solution

Based on the foregoing modeling exercises, the following hypothesisdriven experiments provide verification in chemical and biologicalsystems and further differentiate the method of this invention fromprior art.

Ethyl pyruvate affords a more stable aqueous solution than sodiumpyruvate in the presence of calcium salts (Ringer's solution), and thisobservation can be extended to the study of other pyruvate analogs, asshown in FIG. 1, by dissolving them in Ringer's solution containing atleast 0.2 equivalent of calcium per molar equivalent of pyruvate analogtitrated with sodium hydroxide, or other suitable inorganic alkali, tophysiological pH values. Specifically, the preferred embodiment of this“pyruvated” Ringer's solution for use in NMR, stability, and subsequentbiological studies is shown in Table 2. It is to be understood that thepyruvate analog in the instant example may be substituted with any ofthe analogs shown in FIG. 1 at any concentration sufficient to afford ahomogenous solution or substituted by control substances for comparativepurposes, such as pyruvic acid, lactic acid (as would be the case in“lactated” Ringer's solution and other reference or inactive ketoacidanalogs. The calcium cation may also be substituted, e.g., withmagnesium or any other biologically safe cation capable of substitutingfor calcium and stabilizing the formation of transient coordinationcomplexes with pyruvate ester enolates in aqueous solution. TABLE 2Constituents of Pyruvated Ringer's Solution Component Composition RangeIsotonic saline 75 cc (fixed) KCl 11.25 (fixed) CaCl₂ 7.5 mg 5-20 mgEthyl pyruvate 0.781 ml 0.5-1.5 ml NaOH To pH 7.5 7.35-7.55 (pH)

Following the procedural recommendations for analysis of Margolis et al.(1986) with respect to scanning times and frequencies on a 400 MHzspectrometer operating in pulse-Fourier transform mode, both proton andcarbon shifts in the characteristic resonances for each carbon andproton cluster at the enolizable carbon were monitored as a function oftime and demonstrated that a greater proportion of pyruvate estersshowed a propensity to enolize in Ringer's solutions, especially thosecontaining calcium or magnesium, while pyruvate acid anions showedpreponderant hydration and dimerization under similar conditions. Theultraviolet absorptions of these solutions were also measuredperiodically over the 230-260 nm range and 300-340 nm span, wherechanges in enol formation become evident, and provided confirmatoryevidence about the distinctly different solvation properties of pyruvateester analogs in comparison to pyruvate salts applied in the variousmethods of prior art.

The experimental sequence in which to establish the greater utility ofthe pyruvate derivatives in this invention follows along the same linesas the comparative spectral experiments just described. The samesolutions of test substances used to demonstrate enolization and relatedphenomena were also used in the comparison of basal values for eachcandidate pyruvate to the effects of oxidants on the disappearance ofcharacteristic pyruvate resonances and the appearance of acetate orother degradants of the initial test preparation as a function ofexposure to these oxidants.

For example, 1 mMolar solutions of pyruvic acid and ethyl pyruvateshowed average absorption values, corrected for blanks, of 0.15 and 0.2respectively at 230-260 nm in the absense of calcium at pH 7.2; additionof calcium had no effect on pyruvate, which showed only a marginalincrease in absorption to 0.16, while ethyl pyruvate rose twofold to0.41 in 3 replicate experiments with a coefficient of variation of lessthan 15%. When 28 mM solutions were examined in a similar manner at300-340 nm, the absorbance of pyruvate remained unchanged before andafter calcium addition at a value of 0.03, while the ethyl pyruvatesolutions become noticeably straw colored to the naked eye, rising inabsorbance from 0.07 to 0.85. The yellow coloration and increases inspectrophotometric absorption in the ultraviolet region confirms theformation of a 1,3-conjugated ketone system, as would result from theenolization of ethylpyruvate under conditions which appear not toenolize pyruvic acid.

Thus, applications of hypervalent oxygen mimics, whose redox potentialis known to be a model for ROS, such as hydrogen peroxide, Fenton'sreagent, and meta-chloroperbenzoic acid, were dispensed into the testsolutions at concentrations ranging from 1 to 50 mMolar and theirdegradative effects noted. It was shown that pyruvate esters consume agreater proportion of oxidant per molar equivalent than their congenericfree acid analogs.

EXAMPLE 4 Stability and Reactivity of Pyruvate Esters in Tissue Culture

Pyruvate esters, and in particular ethyl pyruvate, in the presence ofcalcium ion are sufficiently lipophilic to be taken up by cells at afaster rate than equimolar amounts of pyruvate in the cell preparationperfusate. Moreover, the compounds of this invention serve as prodrugsfor intracellular pyruvate delivery and are, therefore, utilized asantioxidants in part by direct decarboxylation of the pyruvate moietythat is delivered intracellularly and made bioavailable afternon-specific ester solvolysis by ubiquitous cytosolic carboxylesterases.Prior to hydrolysis intracellularly, these pyruvate beneficially viaenol-mediated, transient epoxidation mediated by hypervalent oxygen, andrelated toxic oxidants, to form 3-hydropyruvates.

The resulting hydroxypyruvate esters, especially in the case of ethylpyruvate and its analogs which are depicted in FIG. 1, are then taken upas a metabolic fuel by anapleurotic incorporation, after solvolysis, orsubjected to further decarboxylative oxidation by additional equivalentsof reactive oxygen species to form the corresponding hydroxyacetates(glyoxylic acids). Thus, it is to be understood that pyruvate esters canquench twice as many reactive oxygen species than the non-enolyzingforms of the corresponding unesterified ketoacid anion; that is, firstby the formation of 3-hydroxypyruvates and then by the latter'sdecarboxylative degradation into a smaller metabolite, which likeacetate can be readily incorporated into intermediary metabolism. Theseoutcomes in which the compounds of this invention prove more effectiveantioxidants, as well as metabolic fuels, after exposure to ROS aredemonstrable by combinations of NMR and spectral (UV) analyticalprocedures that follow, for example, the fate of stable isotope labeledpyruvate [3-¹³C] species under various experimental conditions.

Accordingly, cell and tissue cultures present a effective means forcomparing the relative rates of uptake and subsequent disposition ofpyruvate analogs dispensed into the culture or perfusion medium and thenmonitored for incorporation into cells by means of a stable isotopictracer that is amenable to proton and carbon magnetic resonance analysisin real time or by mean of mass spectral analysis of suitable extractsof the test biomass after a suitable period of incubation or perfusion.

In particular, since bowel ischemia is one of the more damagingconditions for which pyruvates are known to provide rescue andresuscitation, the use of enterocyte cell cultures provides aappropriate test model. This model consists of exposing enterocytesafter a basal period under various conditions of anoxia and thenhyperoxia to a perfusate containing Ringer's solution supplemented withcalcium as control and then various tests compositions of pyruvates,including sodium pyruvate, all labeled at the 3-methyl position with¹³C. For the carbon MR experiments, cells are seeded on the surface ofpolystyrene microcarrier beads in bacteriological Petri dishes and grownfor 3 days to confluency before harvesting and spectroscopic analysis,following the method of Artemov et al. (1998) and modeling rubrics of Yuet al. (1997) and of Vogt et al. (1997). The test perfusates during thestudy period are also monitored for purposes of background subtractionfrom the acquisition of carbon resonances characteristic of the Krebscycle.

Thus, the rate of carbon flux of exogenously added pyruvate can befollowed throughout the process of conversion into citrate andketoglutarate/glutamic acid. The 3-carbon of pyruvate and the 2-carbonof acetate, derived from pyruvate, are expected to provide differentialenrichments at the 2 versus the 4 position of citrate and ketoglutarate.Direct incorporation of the pyruvate carbon skeleton into citrate andketoglutarate should be expressed as a faster increase in label at the 2position versus the 4 position, since the latter is more likely todiluted by the larger acetate-acetyl-CoA pool.

If hydroxypyruvate is formed in the reaction, not only can the methylgroup resonance be detected directly, but the subsequent utilization ofhydroxypyruvate via decarboxylation into glyoxylate and homologation tomalate can also be traced by the same scheme of differential labelinganalysis. Experiments of this nature confirm that pyruvate esters actdifferently as a carbon source from pyruvate salts. Furthermore, suchexperiments confirm that lactate, acetoacetate and related esters, whensubstituted for pyruvate esters, do not show enolization and are notincorporated into cells and/or processed via oxidative metabolism in amanner similar to, and to the extent of, the pyruvate esters used in themethod of this invention.

EXAMPLE 5 Application of the Invention in Ischemia Rescue

The utility of ethyl pyruvate in a Ringer's solution infusate as aresuscitation fluid in ischemia/reperfusion mucosal injury and barrierdysfunction is demonstrated in this illustrative experiment using a ratmodel of superior mesenteric artery occlusion. The model system andcalculation parameters are illustrated in FIG. 3.

After induction of general anesthesia using intraperitoneal ketamine andpentobarbital, male Sprague-Dawley rats (250-350 g) were subjected to 60minutes of superior mesenteric artery occlusion followed by 60 minutesof reperfusion. Heart rate and mean arterial blood pressure weremeasured via a right carotid arterial catheter. The left internaljugular vein was cannulated for intravenous infusions.

Controls (n=6) received lactated Ringer's solution (lactate, 28 nM,111.5 ml/kg/hr infusion, 1.5 ml/kg bolus prior to ischemia, and 3.0ml/kg bolus prior to reperfusion). Experimental groups (n=6 each)received similar volumes (3 ml) of either pyruvate Na salt (28 mM) orpyruvate ethyl ester (28 mM), prepared in accordance with the method ofthis invention as shown in Table 2 and at a dosage rate equivalent to 10mg/kg/hr. Small intestinal mucosal-to-serosal permeability (CMS,nl/min/cm²) of FITC-dextran (mw=4 kDA) was evaluated using an evertedgut sac technique as previously described by Wattanasirichaigoon (1999).Permeability was measured at baseline, after 30 and 60 minutes ofischemia (130 and 160) respectively, and after 30 and 60 minutes ofreperfusion (R30 and R60, respectively). Histologic samples at baseline,160 and R60 were evaluated for villous height (VH, μ) and mucosalthickness (MT, μ). Mucosal injury grade was determined according to themethod described by Chiu et al. (1970), scored as in Table 3, asfollows: TABLE 3 Mucosal Injury Grade Grade 0 Normal Mucosa Grade 1Subepithelial space formation Grade 2 Moderate epithelial liftingconfined to the tip of the villi Grade 3 Extensive epithelial lifting, afew tips are denuded Grade 4 Denuded villi, dilated exposed capillaries,increased cellularity in the lamina propria Grade 5 Hemorrhagiculceration

Data were summarized as means±standard error of the mean. Significancesof differences were determined using Student's t-test. Differences wereconsidered significant for p<0.05.

The results of these experiments on the utility of the method ofinvention revealed that both pyruvate compositions, as free acid as wellas ethyl ester, significantly decreased mucosal permeability duringreperfusion, as shown in FIG. 4. The ester showed a significant trendtowards effecting earlier and greater cytoprotection as judged by theextent of permeability increase, which is a sign of irreversible tissuedamage and in terms of the significant diminution in mucosal injuryscore, shown graphically in FIG. 5. Pyruvate ethyl ester, moreover,significantly maintained villous height and mucosal thickness duringboth ischemia and reperfusion (p<0.01) as shown in Table 4: TABLE 4Histological Findings on Beneficial Effects of “Pyruvated” Ringer'sSolution Pyruvate Lactate Pyruvate Ester VH MT VH MT VH MT Baseline 470± 30 553 ± 34 461 ± 25 524 ± 28 486 ± 12 583 ± 8 I60 244 ± 20 298 ± 32290 ± 30 372 ± 36 381 ± 24§ 466 ± 25§ R60 130 ± 25 141 ± 22 201 ± 44 266± 50 296 ± 26§ 352 ± 34§Note:Lactate vs Pyruvate and Lactate vs Pyruvate Ester, p < 0.05 and §p <0.01

Taken as a whole, these findings confirm the utility of pyruvate estersin the method of this invention in compositions for the treatment ofischemia and related conditions caused by hypoxia and then reperfusion,with its attendant reactive oxygen damage. The model system describedabove, a rat model of superior mesenteric artery occlusion, is astandard model system familiar to those of ordinary skill who wish toprovide therapeutic treatment of the kind described, and the resultsreported above are easily extrapolatable for human use.

Thus, it is apparent that there has been provided, in accordance withthe invention, novel 2-ketoalkanoic acid ester compounds andcompositions and methods of treating the deleterious effects ofhypervalent oxidants resulting from hypoxic damage, followed byreperfusion, that fully satisfies the objects, aims and advantages setforth above.

While the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives,modifications, and variations will be apparent to those skilled in theart in light of the foregoing description. Accordingly, it is intendedthat the invention shall be directed to all such alternatives,modifications and variations as fall within the spirit and broad scopeof the appended claims.

1. A method for treating a mammal suffering from a conditioncharacterized by ischemia or reperfusion injury, comprisingadministering to said mammal a therapeutically effective amount of acomposition comprising an ester of a 2-ketoalkanoic acid selected fromthe group consisting of alkyl, aralkyl, alkoxyalkyl and carboxyalkylester in a pharmaceutically-acceptable carrier, wherein said carrierfurther comprises a biologically safe component for inducing andstabilizing enolization of the 2-keto functionality of said ester atphysiological pH values.
 2. The method of claim 1, wherein saidpharmaceutically-acceptable carrier is a Ringer's solution of isotonicsaline supplemented with potassium ion.
 3. The method of claim 1,wherein said 2-ketoalkanoic acid ester is selected from the groupconsisting of ethyl pyruvate, propyl pyruvate, butyl pyruvate,carboxymethylpyruvate, acetoxymethylpyruvate, carbethoxymethylpyruvateand ethoxymethylpyruvate.
 4. The method of claim 1, wherein said2-ketoalkanoic acid ester is selected from the group consisting of ethyl2-keto-buyrate, ethyl 2-ketopentanoate, ethyl 2-keto-3-methyl-butyrate,ethyl 2-keto-4-methyl-pentanoate and ethyl 2-keto-hexanoate.
 5. Themethod of claim 3, wherein said 2-ketoalkanoic acid ester is admixed ina saline solution, said solution containing a cation selected from thegroup consisting of calcium and magnesium.
 6. The method of claim 4,wherein said 2-ketoalkanoic acid ester is admixed in a saline solution,said solution containing a cation selected from the group consisting ofcalcium and magnesium.
 7. The method of claim 1, wherein the conditioncharacterized by ischemia is selected from the group consisting ofmesenteric ischemia, mesenteric thrombus, mesenteric venous occlusion,aortic aneurism repair, coronary artery bypass and surgical treatment ofarterial occlusion of limbs.
 8. A process for preserving organ parts,organs or limbs removed from a living mammal, said process comprisingperfusing said organ or limb with a solution containing an effectiveamount of a composition comprising an ester of a 2-ketoalkanoic acidselected from the group consisting of alkyl, aralkyl, alkoxyalkyl andcarboxyalkyl ester in a pharmaceutically-acceptable carrier, saidcarrier further comprising a biologically safe component for inducingand stabilizing enolization of the 2-keto functionality of said ester atphysiological pH values.
 9. The process of claim 8, wherein said2-ketoalkanoic acid ester is selected from the group consisting of ethylpyruvate, propyl pyruvate, butyl pyruvate, carboxymethylpyruvate,acetoxymethylpyruvate, carbethoxymethylpyruvate andethoxymethylpyruvate.
 10. The process of claim 8, wherein said2-ketoalkanoic acid ester is selected from the group consisting of ethyl2-keto-butyrate, ethyl 2-ketopentanoate, ethyl 2-keto-3-methyl-butyrate,ethyl 2-keto-4-methyl-pentanoate and ethyl 2-keto-hexanoate.
 11. Theprocess of claim 9, wherein said 2-ketoalkanoic acid ester is admixed ina saline solution, said solution containing a cation selected from thegroup consisting of calcium and magnesium
 12. The process of claim 10,wherein said 2-ketoalkanoic acid ester is admixed in a saline solution,said solution containing a cation selected from the group consisting ofcalcium and magnesium.
 13. The process of claim 9, wherein said2-ketoalkanoic acid ester is ethyl pyruvate.
 14. The method of claim 1,wherein said 2-ketoalkanoic acid ester is an alkyl ester.
 15. The methodof claim 14, wherein said alkyl ester is ethyl pyruvate.
 16. The methodof claim 1, wherein said condition is characterized by ischemia.
 17. Themethod of claim 16, wherein said ischemia is due to an ischemic eventselected from the group consisting myocardial infarction, cerebralinfarction and intestinal infarction.
 18. The method of claim 1, whereinsaid condition is characterized by reperfusion injury.